Receptor Occupancy of Nonpeptide Corticotropin-Releasing Factor 1 Antagonist DMP696: Correlation with Drug Exposure and Anxiolytic Efficacy

Abstract

4-(1,3-Dimethoxyprop-2-ylamine)-2,7-dimethyl-8-(2,4-dichlorophenyl)-pyrazolo[1,5-a]-1,3,5-triazine (DMP696) is a highly selective and potent, nonpeptide corticotropin-releasing factor 1 (CRF 1 ) antagonist. In this study, we measured in vivo CRF 1 receptor occupancy of DMP696 by using ex vivo ligand binding and quantitative autoradiography and explored the relationship of receptor occupancy with plasma and brain exposure and behavioral efficacy. In vitro affinity (IC 50 ) of DMP696 to brain CRF 1 receptors measured using the brain section binding autoradiography in this study is similar to that assessed using homogenized cell membrane assays previously. The ex vivo binding assay was validated by demonstrating that potential underestimation of receptor occupancy with this procedure could be minimized by identifying an appropriate in vitro incubation time (40 min) based upon the dissociation kinetics of DMP696. Orally administrated DMP696 dose dependently occupied CRF 1 receptors in the brain, with ϳ60% occupancy at 3 mg/kg. In the defensive withdrawal test of anxiety, this dose of DMP696 produced approximately 50% reduction in the exit latency. The time course of plasma and brain drug levels paralleled that of receptor occupancy, with peak exposure at 90 min after dosing. The plasma-free concentration of DMP696 corresponding to 50% CRF 1 receptor occupancy (in vivo IC 50 , 1.22 nM) was similar to the in vitro IC 50 (ϳ1.0 nM). Brain concentrations of DMP696 were over 150-fold higher than the plasma-free levels. In conclusion, doses of DMP696 occupying over 50% brain CRF 1 receptors are consistent with doses producing anxiolytic efficacy in the defense withdrawal test of anxiety, and the IC 50 value estimated in vivo based on plasmafree drug concentrations is consistent with the in vitro IC 50 value.Corticotropin releasing factor (CRF), a 41-amino acid peptide, plays a pivotal role in the behavioral, endocrine, immune, and autonomic responses of the body to stress (Owens and Nemeroff, 1991). In addition to the hypothalamic paraventricular nucleus where it was originally identified, CRF is also widely distributed across brain regions (Chalmers et al., 1996;Heinrichs and De Souza, 1999;Gilligan et al., 2000a). The physiological functions of CRF are mediated by at least two G-protein coupled receptors, CRF 1 and CRF 2 (including splice variants CRF 2␣ , CRF 2␤ , CRF 2␥ ), both of which are linked to adenylyl cyclase activation but have distinct brain distributions. CRF 1 receptors are widespread in the cortex, limbic system, cerebellum, and pituitary, whereas CRF 2 receptors are dominant in subcortical areas including the lateral septum (CRF 2␣ ), ventromedial hypothalamus (CRF 2␣ ), and choroid plexus (CRF 2␤ ) (De Souza, 1987;Chalmers et al., 1995;Primus et al., 1997;Rominger et al., 1998).Increasing evidence suggests that the CRF system is involved in pathophysiology of anxiety disorders (Heinrichs and De Souza, 1999;Gilligan et al., 2000a). Intracerebroventricular administration of CRF induces stress beha...

Prediction of Human Serotonin and Norepinephrine Transporter Occupancy of Duloxetine by Pharmacokinetic/Pharmacodynamic Modeling in the Rat

“…These studies support the strategic use of robust methodologies for the assessment of ex vivo rodent CNS occupancy, and subsequent PK/PD analysis, as a means for the prospective prediction of CNS occupancy in humans. Accurate assessment of transporter (or receptor) occupancy using ex vivo methodology is sensitive to many factors including the temporal effects of tissue processing (Li et al, 2003(Li et al, , 2006. Time-dependent decreases in apparent transporter occupancy have been observed for SERT and NET by using venlafaxine and nomifensine, respectively (Lengyel et al, 2008).…”

PET Imaging of CRF1 with [11C]R121920 and [11C]DMP696: is the target of sufficient density?

“…Methods for in vitro CRF1 binding autoradiography in post-mortem human brain samples were adapted from three reports [9,44,45]. Human and baboon brain sections cut at 20-µm thick were pre-incubated for 30 min at 23° C in 50 mM HEPES buffer (pH 7.2) containing 10 mM MgCl 2 , 2 mM EGTA, 100 mM KIU/ml aprotinin, 0.1 mM bacitracin, 0.1 mM ovalbumin.…”

Anxiolytic-Like Effects of the Corticotropin-Releasing Factor1 (CRF1) Antagonist DMP904 [4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine] Administered Acutely or Chronically at Doses Occupying Central CRF1 Receptors in Rats

“…The procedure described by Li et al (2003) was used. Briefly, following testing in the defensive withdrawal procedure (75 min following oral administration of DMP904), animals were decapitated, and brain tissue was excised, frozen, and sectioned.…”

“…The procedure described by Li et al (2003) was used to determine systemic DMP904 exposure levels immediately after behavioral testing (75 min after oral administration). DMP904 concentration in the plasma was measured using a liquid chromatography tandem mass spectrometric method (LC/MS/ MS).…”